An effective method to extract DNA from environmental samples for polymerase chain reaction amplification and DNA fingerprint analysis

Abstract

A rapid direct-extraction method was used to obtain DNA from environmental soil samples. Heat, enzymes, and guanidine isothiocyanate were utilized to lyse cells. The DNA was purified by agarose gel electrophoresis, amplified with 16S rRNA-based primers by use of the polymerase chain reaction, and then digested with the restriction endonuclease PalI. The extraction method was used to obtain DNA from a variety of plants, bacteria, and fungi including Gossypium hirsucum (cotton), Pseudomonas, Bacillus, Streptomyces, and Colletotrichum. Up to 100 micrograms DNA/g (wet weight) of soil and 400 micrograms DNA/g of plant material were recovered. Restriction endonuclease analysis patterns of amplified rDNA from pure microbial cultures and plant species contained three to five different DNA fragments. Amplified rDNA of mixed population DNA extracts from soil samples, digested with the restriction endonuclease PalI, contained 12-20 DNA fragments, appearing as sample "fingerprints." Results from eight environmental soil samples that were analyzed suggest that the amplified rDNA fingerprints can be used to help characterize the genetic and biological diversity of the microbial populations in these samples.

Phylogenetic Analysis of Anaerobic Psychrophilic Enrichment Cultures Obtained from a Greenland Glacier Ice Core

“…The most comprehensive lysis, as indicated by microscopic examination of the cells, incorporated modifications of the method reported by Porteous et al (27), in which the cells were subjected to bead beater disruption. Turbid anaerobic cultures (total combined volume, 750 ml) were initially filtered by using a 0.2-m-pore-size filter and resuspended in 10 ml of 10 mM Tris, pH 8.0.…”

Tessaracoccus bendigoensis gen. nov., sp. nov., a Gram-positive coccus occurring in regular packages or tetrads, isolated from activated sludge biomass

“…The DNA base composition was calculated from the melting temperature determined by the thermal denaturation method of Owen & Lapage, (1976). Cell DNA was isolated using the method of Porteous et al (1994), modified by disrupting the cells with a cell homogenizer (Braun) and incubating them with 50mg lysozyme ml-l (Sigma) and 100 U achromopeptidase ml-l (Sigma) for 60 min at 37 "C before DNA extraction (Porteous et al, 1994). This method was used to overcome difficulties associated with the production of large amounts of extracellular polysaccharide material by this organism.…”

DNA isolation from soil samples for cloning in different hosts

“…Direct extraction of DNA introduces less bias than methods which are based on cell extraction prior to lysis (von Wintzingerode et al 1997). To extract DNA, the microorganisms in the soil can be lysed mechanically by bead-beating (Moré et al 1994) (Berthelet et al 1996) (Purdy et al 1996), sonication (Degrange and Bardin 1995), freezethawing (Lee et al 1996), grinding in liquid nitrogen (Johnston and Aust 1994) (Volossiouk et al 1995) or enzymatically, using enzymes such as proteinase K (Wikstrom et al 1996;Zhou et al 1996), lysozyme (Porteous et al 1994) or pronase. A combination of these treatments may also be applied (Tsai and Olson 1991) (Tebbe and Vahjen 1993) (Picard et al 1992).…”